Nov 23, 2021
Multi tissue processing for single cell sequencing of human immune cells
- Daniel Rainbow1,
- Sarah Howlett1,
- Lorna Jarvis1,
- Joanne Jones1
- 1Department of Clinical Neuroscience, University of Cambridge, Cambridge, UK
- Cambridge University
Protocol Citation: Daniel Rainbow, Sarah Howlett, Lorna Jarvis, Joanne Jones 2021. Multi tissue processing for single cell sequencing of human immune cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bz4qp8vw
Manuscript citation:
The gut processing protocol has been taken from: James, K.R., Gomes, T., Elmentaite, R. et al. Distinct microbial and immune niches of the human colon. Nat Immunol 21, 343–353 (2020). https://doi.org/10.1038/s41590-020-0602- The skin processing protocol has been taken from: Human skin single cell dissociation on Protocols.io https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 16, 2021
Last Modified: November 23, 2021
Protocol Integer ID: 55152
Keywords: Human, tissue, single cell RNA sequencing, Atlas
Abstract
This protocol has been developed for the simultaneous processing of multiple human tissues to extract immune cells for single cell RNA sequencing using the 10X platform, and ideal for atlasing projects. Included in this protocol are the steps needed to go from tissue to loading the 10X Chromium for single cell RNA sequencing and includes the hashtag and CiteSeq labelling of cells as well as the details needed to stimulate cells with PMA+I.
Attachments
322-685.pdf
366KB
Guidelines
This protocol has been optimised for extracting immune cells from small pieces of tissue (around 10g or less) and has divided tissues into five categories depending on how much mechanical or chemical digestion is needed to enable extraction of immune cells, as shown in the workflow diagram. Blood and bone marrow need no processing. Lymphoid tissues like spleen and lymph nodes require a gentle mashing to make a cell suspension. Non-lymphoid tissues like lung, liver and kidney require both a mechanical and collagenase digestion. The gut and skin require more specialised protocols and use published protocols from the James and Hanifa laboratories.
We include an activation step using PMA+I for 2 hours, however this stimulation condition will need optimising depending on the hypothesis being tested.
Materials
Reagent
X-VIVOTM 15 Serum-free Hematopoietic Cell MediumLonzaCatalog #BE02-060Q
Fetal Bovine SerumMerck MilliporeCatalog #F7524
Dulbecco′s Phosphate Buffered SalineMerck MilliporeCatalog #D8537-500ML
Gibco™ Bovine Albumin Fraction V (7.5% solution)Thermo Fisher ScientificCatalog #15260037
UltraPure 0.5M EDTA, pH 8.0Thermo Fisher ScientificCatalog #15575-038
Roche DTT 14-DithiothreitolSigma AldrichCatalog #10197777001
USB Dithiothreitol (DTT) 0.1M SolutionThermo Fisher ScientificCatalog #707265ML
Collagenase IV, Merck, C7926-100MG
Dispase® II proteaseSigma AldrichCatalog #D4693-1G
Benzonase® Nuclease Purity > 90%Merck MilliporeCatalog #70746-4
Ficoll Paque Plus 500mLGe HealthcareCatalog #17-1440-03
eBioscience™ Cell Stimulation Cocktail (500X)Thermo FisherCatalog #00-4970-93
Hashtags, Biolegend, Total-C
TotalSeq™-C Human Universal Cocktail V1.0BioLegendCatalog #399905
Solutions to make:
| A | B | C | D | |
| Solution | Base | Reagent 1 | Reagent 2 | |
| X-vivo + 1% FBS | 49.5 ml x-vivo | 0.5 ml FBS | 2.5 µl Benzonase | |
| PBS + 0.04% BSA | 500 ml PBS | 2.66 ml | 7.5% BSA | |
| X-vivo +5 mM EDTA + 2 mM DTT + 1% FBS | 48.5 ml x-vivo + 1% FBS | 0.5 ml of 0.5M EDTA | 1 ml of 100 mM DTT |
Equipment
Flowmi® Cell Strainers
NAME
Cell Strainers
TYPE
Flowmi®
BRAND
BAH136800040
SKU
LINK
Equipment
3ml Syringe, disposable, sterile,
NAME
3ml Syringe, disposable, sterile,
TYPE
Terumo
BRAND
GS574
SKU
LINK
Equipment
70 µm Cell Strainer
NAME
Cell Strainer
TYPE
Falcon
BRAND
352350
SKU
LINK
White, Sterile, Individually Packaged
SPECIFICATIONS
Equipment
gentleMACS™ C Tubes
NAME
C Tubes
TYPE
gentleMACS™
BRAND
130-093-237
SKU
LINK
Equipment
In addition to the regular equipment found in a Containment level 2 laboratory you will need:
- Miltenyi Gentlemacs
- 37 °C incubator
- 10X Chromium instrument
Citations
The gut processing protocol has been taken from:
CITATION
The skin processing protocol has been taken from:
Human skin single cell dissociation on Protocols.io https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn
Tissue to cell suspension - Bone Marrow and Blood
Tissue to cell suspension - Bone Marrow and Blood
No processing, go straight to ficoll layering.
PlaceOn ice until other tissues have caught up.
Tissue to cell suspension - Lymphoid Tissues (Spleen, Lymph node)
Tissue to cell suspension - Lymphoid Tissues (Spleen, Lymph node)
Mash the lymphoid tissue through a 70 µm filter placed on top of a 50 mL falcon, using the plunger from a 2 mL syringe as a pestle.
Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.
Depending on the size of the tissue, top up the filtered cell suspension to 30 mL to 50 mL with x-vivo + 1% FBS.
Place On ice until other tissues have caught up.
Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
Tissue to cell suspension - Non-lymphoid tissue (Lung, Liver, Kidney)
32m 22s
32m 22s
We receive around 5 g of tissue and the protocol will need to be scaled up if more tissue is being processed.
Note
Do not overload the Gentlemacs as this will reduce cell yield.
Chop up the tissue with scissors into 0.5 cm pieces.
Do not overload the Gentlemacs C-tube, with no more than 2.5 g of tissue.
Transfer to Gentlemacs tube and add 2.5 mL of collagenase and 2.5 mL x-vivo.
Run the following programme that takes 00:32:00 .
32m
Loop .
- Loop .(1/3)
- Loop. (2/3)
- Loop. (3/3)
Ramp900 rpm 00:00:12 .
12s
Spin 700 rpm, 00:00:01 .
1s
Ramp 1000 rpm, 00:00:08 .
8s
Spin 1500 rpm, 00:00:01 .
1s
Spin 1900 rpm, 00:00:04 .
4s
Spin 1500 rpm, 00:00:01 .
1s
Spin 1900 rpm, 00:00:03 .
3s
Temperature on 37 °C and loop.
- loop (1/2).
- loop (2/2).
Spin 50 rpm, 00:15:00 .
15m
Spin 350 rpm, 00:00:20 .
20s
Add 20 µL of 0.5 millimolar (mM) EDTA ( 2 millimolar (mM) final conc.) per5 mL of collagenase to neutralise and shake to mix.
Pour and scrape digested tissue into a 70 µm cell strainer placed on top of a 50 mL falcon.
Use the plunger of a 2 mL syringe to mash tissue through the filter, like a pestle.
Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.
Depending on the size of the tissue, top up the filtered cell suspension to 30 mL to 50 mL with x-vivo + 1% FBS.
Place On ice until other tissues have caught up.
Tissue to cell suspension - Jejunum
Tissue to cell suspension - Jejunum
1h 32m
1h 32m
Note
Protocol adapted from from Kylie James “Distinct microbial and immune niches of the human colon”, Nature Immunology, 2020. We receive around 5g of tissue and the protocol will need to be scaled up if more tissue is being processed. Do not overload the digestion steps as this will reduce cell yield.
Wash jejunum with PBS + 0.04% BSA to remove any chime.
Chop up the jejunum with scissors into 0.5 cm pieces.
Transfer to a 50 mL falcon tube and add 10 mL of x-vivo + 2 millimolar (mM) DTT +5 millimolar (mM) EDTA + 1% FBS and put in the 37 °C incubator for 00:20:00 and shake after 00:10:00 .
30m
Put jejunum chemical digest through a 70 µm filter on top of a 50 mL falcon and rinse with 10 mL of x-vivo + 1% FCS.
The wash through from the filter contains the IEL cells, keeping the falcon On ice .
Scrape tissue from the filter back into a 50 mL falcon and repeat the digest with 10 mL x-vivo + 2 millimolar (mM) DTT +5 millimolar (mM) EDTA + 1% FBS and place back in the 37 °C incubator for 00:20:00 , and shake after 00:10:00 .
30m
Put jejunum digest through a 70 µm filter on top of the 50 mL falcon containing the IEL cells and rinse with 10 mL of x-vivo + 1% FCS. Keep the IEL cellsOn ice .
Scrape tissue from the filter into a Gentlemacs C tube and digest with 2.5 mL of collagenase and 2.5 mL of x-vivo and run the programme called ‘Sarah’ takes00:32:00 , with various mixing speeds.
32m
Add 20 µL of 0.5 millimolar (mM) EDTA ( 2 millimolar (mM) final conc) per 5 mL of collagenase to neutralise and shake to mix.
Pour and scrape digested tissue into a 70 µm cell strainer placed on top of a 50 mL falcon.
Use the plunger of a 2 mL syringe to mash tissue through the filter, like a pestle.
Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue, cells that pass through the filter are LP cells.
Depending on the size of the tissue, top up the filtered cell suspension to 30 mL to 50 mL with x-vivo + 1% FBS.
Place On ice until other tissues have caught up.
Tissue to cell suspension - Skin
Tissue to cell suspension - Skin
1h 32m
1h 32m
Protocol from Haniffa Lab, Newcastle University https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn
Note
Depending on when tissues arrive, skin can either be set up the night before and then will be processed with all the other tissues. Or will have to be processed the next day.
● If processed the same day as other tissues then will be hashtagged with all other tissues.
● If processed the next day will have to follow the same procedure but the unstim will wait on ice until the stim catches up and can be loaded on 1 lane of 10x.
○ 5 µL of CITE-Seq will need to be left from the processing of the other tissues.
Chop into ~0.5 cm2 sized pieces. Remove as much dermis from each as possible using a razor blade - be careful, extremely sharp. Discard the dermis layer.
Incubate the retained skin in dispase for 02:00:00 to 03:00:00 at 37 °C , to allow the epidermis to be stripped.
5h
Separate the epidermis from the dermis using fine forceps. These can be kept separate or processed together.
Wash in PBS.
Add collagenase at 3X the volume of the tissue and incubate at 37 °C Overnight .
3h
Add 20 µL of 0.5 millimolar (mM) EDTA (2 millimolar (mM) final conc) per 5 mL of collagenase to neutralise and shake to mix.
Scrape the digested skin and media into a70 µm filter on top of a 50 mL falcon.
Use the plunger from a2 mL syringe to mash the skin through the filter, like a pestle.
Occasionally wash the filter with x-vivo + 1% FBS as you mash the tissue.
Depending on the size of the tissue, top up the filtered cell suspension to 30 mL to 50 mL with x-vivo + 1% FBS.
Place On ice until other tissues have caught up, or if processing the next day alone proceed with cell count and ficoll.
Cell suspension to MNC - Wash cell suspension
Cell suspension to MNC - Wash cell suspension
10m
10m
Once all the tissues have reached a cell suspension, spin at 600 x g, 00:10:00 .
10m
Pour off supernatant and resuspend in x-vivo + 1% FBS, the volume to resuspend depends what you are going to layer over ficoll.
There is no exact science to the layering but as a guide:
a. Spleen - 90 mL
b. Lymph nodes - 7 mL
c. Non-lymphoid tissue - up to60 mL
d. Skin - 7 mL
Cell suspension to MNC - Ficoll
Cell suspension to MNC - Ficoll
1h 5m
1h 5m
Note
Number of ficoll tubes to be used depends on the size of the tissue and the cell pellet.
Bone marrow 10 mL + 20 mL x-vivo layer on 15 mL ficoll per 50 mL falcon.
Blood up to 15 mL +15 mL x-vivo layer on 15 mL ficoll per 50 mL falcon.
Spleen 30 mL cells suspension over 15 mL ficoll per 50 mL falcon x3.
Lymph nodes 7 mL cells suspension over 8 mL ficoll per 15 mL falcon.
Non-lymphoid tissue depending on the size of the cell pellet up to30 mL cell suspension over 5 mL ficoll x2.
Skin 7 mL cells suspension over8 mL ficoll per 15 mL falcon.
Spin tubes at 400 x g, 00:25:00 with slow deceleration. Takes around 00:40:00 to run.
1h 5m
Cell suspension to MNC - CD66b and RBC depletion
Cell suspension to MNC - CD66b and RBC depletion
16m 30s
16m 30s
Note
- It is good to remove granulocytes (expressing CD66b) and RBC from each sample where required as these cells do not provide useful single cell sequencing information.
- Use Stem Cell CD66b positive selection kit (17882) to remove granulocytes from each sample and Stemcell RBC depletion reagent (18170).
In a 15 mL falcon, add from0.5 mL to 3 mL of sample (up to 5 million cells).
Add 25 µL of CD66b positive selection cocktail, mix and incubate for 00:03:00 at Room temperature .
3m
Vortex RapidSpheres for 00:00:30 .
30s
Add 25 µL of RapidSpheres, mix and incubate for 00:03:00 at Room temperature .
3m
Add25 µL of RBC depletion reagent per 1 mL of sample and mix.
Immediately place the samples on a magnet for 00:05:00 .
5m
Carefully pipette off the supernatant to a fresh tube and place On ice .
Note
This contains the cells you want, DO NOT discard.
Wash the beads with 5 mL of PBS 1% FBS + 1 millimolar (mM) EDTA and place back on the magnet for 00:05:00 .
5m
Collect supernatant and add to the fresh tube in step 60.
Throw away the leftover tube with beads as this contains the granulocytes and RBC.
Cell suspension to MNC - Count Cells
Cell suspension to MNC - Count Cells
16m 30s
16m 30s
Count cells from each tissue after ficoll (and CD66b / RBC depletion).
Make sure cells are well mixed and count with trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x dilution factor x volume x 10,000 = Total cell count.
Hashtag, CITE-Seq and stimulation - Hashtag
Hashtag, CITE-Seq and stimulation - Hashtag
1h 5m
1h 5m
Take at least 500k MNC per tissue (use 750k to 1 million cells if available) into a1.5 mL lo-bind eppendorf.
Spin cells at 600 x g, 00:05:00 , remove as much supernatant as possible and resuspend in 50 µL PBS+0.04% BSA.
5m
Record which hashtag is used for which tissue.
Add 5 µL FC block and incubate at 4 °C for 00:10:00 .
10m
Spin each hashtag at 14000 x g, 00:10:00 .
10m
Add 0.5 µL of hashtag to each tube.
Incubate at 4 °C for 00:30:00 .
30m
Make up lyophilised CITE-Seq antibodies (see - section CITE-Seq section)
Top up to 500 µL with PBS + 0.04% BSA, and spin at 600 x g, 00:05:00 , and remove supernatant.
5m
Wash cells with 500 µL with PBS + 0.04% BSA, and spin at 600 x g, 00:05:00 , and remove supernatant.
5m
Resuspend in 100 µL of PBS + 0.04% BSA.
Hashtag, CITE-Seq and stimulation - Count cells
Hashtag, CITE-Seq and stimulation - Count cells
1h 5m
1h 5m
Count cells from each tissue after the Hashtag washes as there will be cell loss, and if a particular tissue has fewer cells than needed, then repeat the hashtag process with more cells.
Make sure cells are well mixed and count with trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x dilution factor x volume x 10,000 = Total cell count.
Hashtag, CITE-Seq and stimulation - Pool MNC from all tissues and split for Unstim and Stim (if required)
Hashtag, CITE-Seq and stimulation - Pool MNC from all tissues and split for Unstim and Stim (if required)
5m
5m
Use the post hashtag cell counts to pool MNC from each tissue at equal cell number, based on what the lowest count is, into a 1.5 mL lo-bind eppendorf.
Ideally you want 300k - 400k from each tissue. Record the total volume.
Flick to mix the cells really well.
Remove ⅓ of the cell volume to a new 1.5 mL tube and label as Unstim and top up to 500 µL with PBS + 0.04% BSA. Spin at600 x g, 00:05:00 and proceed to the CITE-Seq section.
5m
To the remaining ⅔ of pooled MNC, label the tube as Stim and top up to 1 mL with x-vivo + 1% FCS and proceed to MNC stimulation.
Hashtag, CITE-Seq and stimulation - MNC Stimulation with PMA+I
Hashtag, CITE-Seq and stimulation - MNC Stimulation with PMA+I
3h 10m
3h 10m
Note
- We are using a PMA+I stimulation which we have optimised to assess early activation events, and depending on the hypothesis being tested may need to be a different stimulant and time point.
Get the MNC stim on as it takes02:00:00 .
2h
Pool culture in MNC in1 mL of x-vivo + 1% FBS for 02:00:00 at 37 °C with 2 µL of cell stim cocktail (PMA+I). Flick tube to mix cells every 00:30:00 to 00:40:00 .
3h 10m
Culture MNC at no more than 2 million cells per ml.
Note
- Use more than one 1.5 mL tube if needed.
- Cell stim cocktail (PMA+I, eBioscience) is 1:500 stock.
Incubate for 02:00:00 , move to Cite-Seq of stimulated cells.
2h
Hashtag, CITE-Seq and stimulation - CITE-Seq
Hashtag, CITE-Seq and stimulation - CITE-Seq
3h 10m
3h 10m
Make up lyophilized CITE-Seq antibodies - each vial is enough for 500k cells, but will use 1 vial for up to 2 million cells.
Spin lyophilised reagent at 10000 x g, 00:00:30 .
30s
Add 27.5 µL Cell staining buffer to the lyophilised CITE-Seq reagent and briefly vortex.
Incubate at Room temperature for 00:05:00 .
5m
Briefly vortex again, then spin at 10000 x g, 00:00:30 .
30s
Transfer entire volume to a lo-bind PROTEIN tube.
Spin at 14000 x g, 4°C, 00:10:00 .
10m
Store in the fridge until ready to use.
Spin the unstim pool MNC at 600 x g, 00:05:00 and remove supernatant.
5m
Resuspend cells in 50 µL of PBS + 0.04% BSA.
No need to add FC block, as already done at hashtag stage.
If not hashtagged already, then add 5 µL FC block for 00:10:00 at 4 °C .
10m
Add10 µL of CITE-Seq 130Ab and incubate at 4 °C for 00:30:00 .
Note
Keep the remaining CITE-Seq reagent for the stimulated sample and the skin if processed the next day.
30m
(Take 10x reagent out of the freezer to warm up to Room temperature , during CITE-Seq incubation. It takes 00:30:00 to warm up to Room temperature .)
30m
Top up to500 µL with PBS + 0.04% BSA, and spin at 600 x g, 00:05:00 , and remove supernatant.
5m
Wash cells with 500 µL with PBS + 0.04% BSA, and spin at 600 x g, 00:05:00 , and remove supernatant.
5m
Resuspend cells in 250 µL PBS + 0.04% BSA and put through a flowmi filter. Rinse out 1.5 mL tube with 250 µL PBS + 0.04% BSA, and put this through the same Flowmi filter.
Note
TIP - use a second 1 mL pipette tip, so can keep Flowmi filter on original tip, remove from pipette and pipette second 250 µL wash into the top of the tip with the filter. Reattach the pipette and wash through the filter.
Spin at 600 x g, 00:05:00 , and remove supernatant.
5m
Resuspend in 100 µL of PBS + 0.04% BSA.
Hashtag, CITE-Seq and stimulation - Count cells
Hashtag, CITE-Seq and stimulation - Count cells
3h 10m
3h 10m
Count the unstim pooedl MNC sample.
Make sure cells are well mixed and count with trypan blue.
2 µL of cells to 8 µL of Trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x 5 x 0.1 x 10,000 = Total cell count.
Hashtag, CITE-Seq and stimulation - Load unstim for 10x
Hashtag, CITE-Seq and stimulation - Load unstim for 10x
Load cells at 1,000 cells per 1 µL (Max 2,000 cells / µl).
Dilute the sample (if needed).
Load 15,000 cells per tissue, 30,000 cells per 10x GEM reaction.
Note
So for 6 tissues, it would be 90,000 cells over 3 10x GEMs.
Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
Hashtag, CITE-Seq and stimulation - Cite-Seq of stimulated cells
3h 20m
3h 20m
After the 02:00:00 stimulation, spin the stim pool MNC at 600 x g, 00:05:00 and remove supernatant.
2h 5m
Resuspend cells in 15 µL of PBS + 0.04% BSA.
Note
No need to add FC block as already done at hashtag stage.
Add 12.5 µL of CITE-Seq 130Ab and incubate at 4 °C for 00:30:00 .
30m
(Take 10x reagent out of the freezer to warm up toRoom temperature , during CITE-Seq incubation. It takes 00:30:00 to warm up toRoom temperature .)
30m
Top up to 500 µL with PBS + 0.04% BSA, and spin at 600 x g, 00:05:00 , and remove supernatant.
5m
Wash cells with 500 µL with PBS + 0.04% BSA, and spin at 600 rpm, 00:05:00 , and remove supernatant.
5m
Resuspend cells in 250 µL PBS + 0.04% BSA and put through a flowmi filter. Rinse out 1.5 mL tube with 250 µL PBS + 0.04% BSA, and put this through the same Flowmi filter.
Note
TIP - use a second 1ml pipette tip, so you can keep Flowmi filter on the original tip, remove from pipette and pipette second 250 µL wash into the top of the tip with the filter. Reattach the pipette and wash through the filter.
Spin at 600 x g, 00:05:00 , and remove supernatant.
5m
Resuspend in 100 µL of PBS + 0.04% BSA.
Hashtag, CITE-Seq and stimulation - Count cells
Hashtag, CITE-Seq and stimulation - Count cells
Count the Stim pooled MNC sample.
Make sure cells are well mixed and count with trypan blue.
2 µL of cells to 8 µL of Trypan blue. If count all 25 squares of the haemocytometer, then:
Cell count x 5 x 0.1 x 10,000 = Total cell count.
Hashtag, CITE-Seq and stimulation - Load stim for 10x
Hashtag, CITE-Seq and stimulation - Load stim for 10x
Load cells at 1,000 cells per 1 µL (Max 2,000 cells / µl).
Dilute the sample (if needed) in PBS + 0.04% BSA.
Load 15,000 cells per tissue, 30,000 cells per 10x GEM reaction.
Note
So for 6 tissues, it would be 90,000 cells over 3 10x GEMs.
Flow, Freezing and RNA from remaining cells - Remaining cells put in RLT
Flow, Freezing and RNA from remaining cells - Remaining cells put in RLT
5m
5m
When all the 10x GEMs have been processed and they look good, pellet any leftover pooled unstim or stim MNC at 600 rpm, 00:05:00 and take off supernatant.
5m
Flick to resuspend dry pellet and resuspend in 350 µL of Qiagen RLT buffer.
Quickly vortex and freeze at -80 °C until ready to extract the RNA.
Flow, Freezing and RNA from remaining cells - Flow cytometry
Flow, Freezing and RNA from remaining cells - Flow cytometry
Run a flow panel to QC the sample and get proportions of the major cell types. Stain ~500k per tissue with the desired panel of antibodies.
Note
This is an example flow cytometry panel, however may need to be adjusted depending on the flow cytometer available:
a. CD3 - Percp Cy5.5
b. CD19 - APC
c. CD56 - PE 10
d. CD4 - PE Cy7
e. CD14 - FITC
f. CD16 - BV421
g. CD8-APCCy7
Fix cells and store at 4 °C until they can be analysed.
Flow, Freezing and RNA from remaining cells - Freeze down excess cells
Flow, Freezing and RNA from remaining cells - Freeze down excess cells
35m
35m
Note
Any cells that are not going for 10x or flow cytometry can be frozen down.
Spin cells at 600 x g, 00:05:00 , and remove as much supernatant as possible.
5m
Flick to resuspend cell pellet.
Add cell freezing media dropwise, until ~ 10 million cells per ml.
Flick to mix, and transfer to labelled NUNC tubes.
Put NUNC tubes in a Mr Frosty and store at-80 °C Overnight .
30m
Next day, transfer to LN2 storage.
Citations
James, K.R., Gomes, T., Elmentaite, R. et al.. Distinct microbial and immune niches of the human colon
10.1038/s41590-020-0602