Staining of cells for flow cytometry: Difference between revisions

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Created page with "{| class="wikitable" |+ ! ! ! ! |- |FACS buffer: |1xPBS FCS (heat-inactivated, final concentration: 5%) 5% Natriumazid (final concentration: 0.1%) | | |- |1st antibody: |primary antibody (approx. dilution 1:200) | | |- |2nd antibody: |secondary antibody (approx. dilution 1:500) fluorescence- labelled | | |- |controls: |sample with irrelevant primary antibody and secondary antibody sample with secondary antibody only if possible untransfected cells stained with both antib..."
 
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{| class="wikitable"
{| class="wikitable"
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!Materials
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FCS (heat-inactivated, final concentration: 5%)
FCS (heat-inactivated, final concentration: 5%)
5% Natriumazid (final concentration: 0.1%)
5% Natriumazid (final concentration: 0.1%)
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|-
|-
|1st antibody:
|1st antibody:
|primary antibody (approx. dilution 1:200)
|primary antibody (approx. dilution 1:200)
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|-
|-
|2nd antibody:
|2nd antibody:
|secondary antibody (approx. dilution 1:500)
|secondary antibody (approx. dilution 1:500)
fluorescence- labelled
fluorescence- labelled
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|-
|-
|controls:
|controls:
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sample with secondary antibody only
sample with secondary antibody only
if possible untransfected cells stained with both antibodies
if possible untransfected cells stained with both antibodies
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|}


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Preparation of cells:
Preparation of cells:


§   aspirate medium and wash twice with 5-10 mL 1xPBS
* aspirate medium and wash twice with 5-10 mL 1xPBS
 
* detach cells with 1 mL Trypsin/EDTA
§   detach cells with 1 mL Trypsin/EDTA
* inactivate Trypsin/EDTA with 3 mL fresh medium
 
* transfer the cells into a 15 mL centrifuge tube and centrifuge 3 min at 600 rpm
§   inactivate Trypsin/EDTA with 3 mL fresh medium
* discard supernatant and resuspend the cell pellet in FACS buffer
 
* centrifuge again, resuspend cells in FACS buffer and determine cell count
§   transfer the cells into a 15 mL centrifuge tube and centrifuge 3 min at 600 rpm
* distribute into Eppendorf tubes (106 cells/tube)
 
§   discard supernatant and resuspend the cell pellet in FACS buffer
 
§   centrifuge again, resuspend cells in FACS buffer and determine cell count
 
§   distribute into Eppendorf tubes (106 cells/tube)
 
 
Staining (perform all steps on ice):
 
§   pellet the cells by centrifugation 2 min, 2500 rpm, 4°C
 
§   resuspend pelled in 300 µL FACS buffer
 
§   add 1st antibody at desired concentration
 
§   incubate 1-1.5h on ice, centrifuge 2 min, 2500 rpm, 4°C
 
§   discard supernatant and resuspend the pellet in 600 µL FACS buffer, centrifuge again as above
 
§   wash 3 times with 300 µL FACS buffer, at the end resuspend in 300 µL FACS buffer
 
§   add 2nd antibody at desired concentration
 
§   incubate max. 30 min on ice (keep samples dark!)
 
§   wash twice with 600 µL FACS buffer
 
§   during centrifugation prepare FACS tubes with 600 µL FACS buffer


§   resuspend pellet in 600 µL FACS buffer and transfer to prepared FACS tubes
=== Staining (perform all steps on ice): ===


§   measure samples
* pellet the cells by centrifugation 2 min, 2500 rpm, 4°C
* resuspend pelled in 300 µL FACS buffer
* add 1st antibody at desired concentration
* incubate 1-1.5h on ice, centrifuge 2 min, 2500 rpm, 4°C
* discard supernatant and resuspend the pellet in 600 µL FACS buffer, centrifuge again as above
* wash 3 times with 300 µL FACS buffer, at the end resuspend in 300 µL FACS buffer
* add 2nd antibody at desired concentration
* incubate max. 30 min on ice (keep samples dark!)
* wash twice with 600 µL FACS buffer
* during centrifugation prepare FACS tubes with 600 µL FACS buffer
* resuspend pellet in 600 µL FACS buffer and transfer to prepared FACS tubes
* measure samples

Latest revision as of 15:00, 20 May 2025

Materials
FACS buffer: 1xPBS

FCS (heat-inactivated, final concentration: 5%) 5% Natriumazid (final concentration: 0.1%)

1st antibody: primary antibody (approx. dilution 1:200)
2nd antibody: secondary antibody (approx. dilution 1:500)

fluorescence- labelled

controls: sample with irrelevant primary antibody and secondary antibody

sample with secondary antibody only if possible untransfected cells stained with both antibodies

Procedure

Preparation of cells:

  • aspirate medium and wash twice with 5-10 mL 1xPBS
  • detach cells with 1 mL Trypsin/EDTA
  • inactivate Trypsin/EDTA with 3 mL fresh medium
  • transfer the cells into a 15 mL centrifuge tube and centrifuge 3 min at 600 rpm
  • discard supernatant and resuspend the cell pellet in FACS buffer
  • centrifuge again, resuspend cells in FACS buffer and determine cell count
  • distribute into Eppendorf tubes (106 cells/tube)

Staining (perform all steps on ice):

  • pellet the cells by centrifugation 2 min, 2500 rpm, 4°C
  • resuspend pelled in 300 µL FACS buffer
  • add 1st antibody at desired concentration
  • incubate 1-1.5h on ice, centrifuge 2 min, 2500 rpm, 4°C
  • discard supernatant and resuspend the pellet in 600 µL FACS buffer, centrifuge again as above
  • wash 3 times with 300 µL FACS buffer, at the end resuspend in 300 µL FACS buffer
  • add 2nd antibody at desired concentration
  • incubate max. 30 min on ice (keep samples dark!)
  • wash twice with 600 µL FACS buffer
  • during centrifugation prepare FACS tubes with 600 µL FACS buffer
  • resuspend pellet in 600 µL FACS buffer and transfer to prepared FACS tubes
  • measure samples
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